Essential Bioinformatics Resources for Designing PCR Primers
and Oligos for Various Applications
Please complete the workshop sign-in form.
Essential Bioinformatics Resources
for Designing PCR Primers and
Oligos for Various Applications
Yi-Bu Chen, Ph.D.
Bioinformatics Specialist
Norris Medical Library
University of Southern California
323-442-3309
yibuchen@belen.hsc.usc.edu
Workshop Outline
A. The General Rules for PCR Primer Design
B. Resources for General Purpose PCR Primer Design
C. Resources for Real-Time q-PCR Primer Design
D. Resources for Site-Directed Mutagenesis PCR Primer Design
E. Resources for PCR Primers/Oligos Quality Analysis
F. Resources for Multiplex PCR Primer Design
G. Resources for Microarray Probes Design
H. Resources for SNPs and Genotyping PCR Applications
I.
Resources for Degenerate PCR Primer Design
J.
Resources Methylation PCR Primer Design
PCR: the technology that changed the world we knew
 The Polymerase Chain Reaction (PCR) revolutionized life
sciences as it provides a sensitive, reliable, efficient, and
convenient means of amplifying relatively large quantities of
DNA
 Invented in 1983 by Kary Mullis, who won a Nobel Prize 1993
 The technique was made possible by the discovery of Taq
polymerase, the DNA polymerase that is used by the bacterium
Thermus aquaticus, discovered in hot springs.
 The primary materials used in PCR:
- DNA nucleotides: the building blocks for the new DNA
- Template DNA: the DNA sequence that you want to amplify
- Primers: single-stranded short DNA (16--50 nucleotides
long) that are complementary to a short region on either end of
the template DNA
- DNA polymerase: a heat stable enzyme that catalyzes the
synthesis of new DNA
Primers dictate the successfulness of a PCR
Specificity?
Proper
annealing to
the template?
Before you design your own primers –
Don’t reinvent the wheels!
Before you start designing primers –
Find and use the right resources!
What are the primers for?
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General purpose amplification?
SNPs detection/validation?
Methylation study?
Real-time PCR?
Microarray probes?
Degenerate PCR?
Multiplex PCR?
What do you have to begin with?



Single DNA/protein sequence?
Multiple DNA/protein sequence files?
GenBank ID/Gene ID/Gene Symbol/rsSNP ID?
After you have your primers designed –
Consider a second opinion!
Most likely your primers can be designed by
several different software
Different software may vary significantly in:



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
Concepts and overall approaches
Designing criteria and default settings
Comprehensiveness
Usability
Accessibility and speed
Consider a second opinion when


You are new to such design task/application
You don’t have a lot of confidence in the initial result
General rules for primer design
-- Primer and amplicon length
Primer length determines the specificity and
significantly affect its annealing to the template


Too short -- low specificity, resulting in non-specific
amplification
Too long -- decrease the template-binding efficiency at
normal annealing temperature due to the higher probability
of forming secondary structures such as hairpins.
Optimal primer length


18-24 bp for general applications
30-35 bp for multiplex PCR
Optimal amplicon size
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300-1000 bp for general application, avoid > 3 kb
50-150 bp for real-time PCR, avoid > 400 bp
General rules for primer design
-- Melting temperature (Tm)
 Tm is the temperature at which 50% of the DNA duplex
dissociates to become single stranded



Determined by primer length, base composition and concentration.
Also affected by the salt concentration of the PCR reaction mix
Working approximation: Tm=2(A+T)+4(G+C) (suitable only for 18mer
or shorter).
 Optimal melting temperature


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52°C-- 60°C
Tm above 65°C should be generally avoided because of the potential for
secondary annealing.
Higher Tm (75°C-- 80°C) is recommended for amplifying high GC
content targets.
 Primer pair Tm mismatch


Significant primer pair Tm mismatch can lead to poor amplification
Desirable Tm difference < 5°C between the primer pair
General rules for primer design
-- Specificity and cross homology
 Specificity


Determined primarily by primer length as well as sequence
The adequacy of primer specificity is dependent on the nature of the
template used in the PCR reaction.
 Cross homology


Cross homology may become a problem when PCR template is genomic
DNA or consists of mixed gene fragments.
Primers containing highly repetitive sequence are prone to generate nonspecific amplicons when amplifying genomic DNA.
 Avoid non-specific amplification



BLASTing PCR primers against NCBI non-redundant sequence database
is a common way to avoid designing primers that may amplify nontargeted homologous regions.
Primers spanning intron-exon boundaries to avoid non-specific
amplification of gDNA due to cDNA contamination.
Primers spanning exon-exon boundaries to avoid non-specific
amplification cDNA due to gDNA contamination.
General rules for primer design
-- GC content; repeats and runs
Primer G/C content
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Optimal G/C content: 45-55%
Common G/C content range: 40-60%
Runs (single base stretches)
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
Long runs increases mis-priming (non-specific annealing)
potential
The maximum acceptable number of runs is 4 bp
Repeats (consecutive di-nucleotide)


Repeats increases mis-priming potential
The maximum acceptable number of repeats is 4 dinucleotide
General rules for primer design
-- Primer secondary structures
 Hairpins


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Formed via intra-molecular interactions
Negatively affect primer-template binding, leading to poor or no
amplification
Acceptable ΔG (free energy required to break the structure): >-2
kcal/mol for 3’end hairpin; >-3 kcal/mol for internal hairpin;
 Self-Dimer (homodimer)


Formed by inter-molecular interactions between the two same primers
Acceptable ΔG: >-5 kcal/mol for 3’end self-dimer; >-6 kcal/mol for
internal self-dimer;
 Cross-Dimer (heterodimer)


Formed by inter-molecular interactions between the sense and antisense
primers
Acceptable ΔG: >-5 kcal/mol for 3’end cross-dimer; >-6 kcal/mol for
internal cross-dimer;
General rules for primer design
-- GC clamp and max 3’ end stability
GC clamp
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Refers to the presence of G or C within the last 4 bases from
the 3’ end of primers
Essential for preventing mis-priming and enhancing specific
primer-template binding
Avoid >3 G’s or C’s near the 3’ end
Max 3’end stability
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

Refers to the maximum ΔG of the 5 bases from the 3’end of
primers.
While higher 3’end stability improves priming efficiency, too
higher stability could negatively affect specificity because of
3’-terminal partial hybridization induced non-specific
extension.
Avoid ΔG < -9.
General rules for primer design
-- Annealing temperatures and other considerations
Ta (Annealing temperature) vs. Tm
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

Ta is determined by the Tm of both primers and amplicons:
optimal Ta=0.3 x Tm(primer)+0.7 x Tm(product)-25
General rule: Ta is 5°C lower than Tm
Higher Ta enhances specific amplification but may lower yields
Crucial in detecting polymorphisms
Primer location on template


Dictated by the purpose of the experiment
For detection purpose, section towards 3’ end may be preferred.
When using composite primers


Initial calculations and considerations should emphasize on the templatespecific part of the primers
Consider nested PCR
http://www.hsls.pitt.edu/guides/genetics/obrc
http://www.usc.edu/hsc/nml/lib-services/bioinformatics/index.html
http://search.hsls.pitt.edu/vivisimo/cgi-bin/query-meta?input-form=molbio-simple&query=pcr+primer&v%3Asources=OBRC&v%3Aproject=molbio
Resources for General Purpose PCR Primer Design
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Primer3
Primer3Plus
PrimerZ
PerlPrimer
Vector NTI Advantage 10
General Purpose PCR Primer Design Tool– Primer3
Name
Type
Key Functions
Publication Info
Times Cited
Pros
Cons
Note
YiBu’s Rating
Primer3 -- an online tool for PCR primer design
Web-based software
Design PCR primers and hybridization probes.
Methods Mol Biol 2000
823
The original and most widely used PCR primer design program; uses sequence
as input; a huge number of options for customizing primer design;
busy interface;
In OBRC; the program has been widely adopted by many primer design
software.
4 out of 5
Web Site:
http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi
More Info:
http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1043858198/info
General Purpose PCR Primer Design Tool– Primer3Plus
Name
Type
Key Functions
Publication Info
Times Cited
Pros
Cons
Note
YiBu’s Rating
Primer3Plus, an enhanced web interface to Primer3
Web-based software
Design PCR primers for a given DNA sequence.
NAR 2007
N/A
Uses sequences or sequence file as input; a huge number of configuration
options; automates specific tasks such as designing primers for cloning or stepwise sequencing; primers can be sent to an order form; clean, intuitive and well
organized interface;
in OBRC. It is an updated, task-oriented web-interface to the original Primer3.
4.5 out of 5
Web Site:
http://www.bioinformatics.nl/primer3plus
More Info:
http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1191263055/info
General Purpose PCR Primer Design Tool– PrimerZ
Name
Type
Key Functions
Publication Info
Times Cited
Pros
Cons
Note
YiBu’s Rating
PrimerZ -- streamlined primer design for promoters, exons and
human/mouse SNPs
Web-based software
Design PCR primers for promoters, exons, and human/mouse SNPs
Nucleic Acid Research 2007
0 (too new)
Uses gene name, Ensembl ID, rs# as input; settings for amplicon region and
length, as well as PCR product sizes; allow batch rsSNP processing; frequently
updated; offers many advance design settings; interactive results output
reported successful rate over 70%; only for human and mouse
built on Primer3; in OBRC.
4.5 out of 5
Web Site:
http://genepipe.ngc.sinica.edu.tw/primerz/beginDesign.do
More Info:
http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1190992855/info
General Purpose PCR Primer Design Tool –
PerlPrimer
Name
Type
Key Functions
Publication Info
Times Cited
Pros
Cons
Note
YiBu’s Rating
PerlPrimer -- cross-platform, graphical primer design for standard,
bisulphite and real-time PCR
Desktop software (for Windows, Linux and Mac OS-X)
Design primers for standard, bisulphite and real-time PCR, and sequencing.
Bioinformatics 2004
8
Cross-platform; versatile applications; retrieving sequences from Ensembl as
input; QPCR primer design without manual intron-exon boundary entry;
BLAST search primers; primer pair quality analysis; ORF and CpG island
detection;
Requires local installation of the software; automatic sequence retrieval only
through Esemble (selected eukaryotic genomes) not NCBI (more
comprehensive).
In OBRC
4 out of 5
Web Site:
http://perlprimer.sourceforge.net/index.html
PerlPrimer screenshots:
http://perlprimer.sourceforge.net/screenshots.html
More Info:
http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1167845497/info
General Purpose PCR Primer Design Tool–
Vector NTI Advance 10
Name
Type
Key Functions
Publication Info
Times Cited
Pros
Cons
Note
YiBu’s Rating
Vector NTI Advance 10 -- design primers and oligos for regular/multiplex
PCR, sequencing, hybridization.
Commercial Desktop software (free to nonprofit users)
Design PCR primers and oligos for routine molecular applications
N/A
N/A
Uses user sequence, multiple DNA sequence alignment as input; commercial
quality features/functions; integrated with other Vector NTI applications; many
design settings; analyzes and ranks primer quality;
requires software installation/licensing;
NML offers workshop and tutorials; in OBRC.
4 out of 5
Web Site for NML Workshop:
http://www.usc.edu/hsc/nml/libservices/bioinformatics/vector_nti_advance_10_workshop.html
More Info On Vector NTI Advance 10:
http://www.usc.edu/hsc/nml/lib-services/bioinformatics/vector_nti_advance_10.html
Primer Design Resources for Real-time PCR
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NCBI Probe Database
RTPrimerDB
Primer Bank
qPrimerDepot
PCR-QPPD
PerlPrimer
Public PCR Primers/Oligo Probes Repository
– The NCBI Probe Database
Name
Type
Key Functions
Publication Info
Times Cited
Pros
Cons
Note
YiBu’s Rating
NCBI Probe Database
Web-based database
Search for documented PCR primers and oligo probes for genotyping, gene
expression, SNP discovery, gene silencing and genome mapping applications.
Unpublished
n/a
The largest database of its kind; results including information on reagent
distributors, probe effectiveness, and computed sequence similarities; Entrez
allows search to be limited by applications, probe type, and model organism;
n/a
4.5 out of 5
Web Site:
Database Overview:
http://www.ncbi.nlm.nih.gov/genome/probe/doc/Overview.shtml
Database Query Tips:
http://www.ncbi.nlm.nih.gov/genome/probe/doc/QueryTips.shtml
http://www.ncbi.nlm.nih.gov/sites/entrez?db=probe
Resources for real time PCR– RTPrimerDB
Name
Type
Key Functions
Publication Info
Times Cited
Pros
Cons
Note
YiBu’s Rating
RTPrimerDB: the real-time PCR primer and probe database
Database
Search for validated primers and probes used in real-time PCR assays
employing popular chemistries (SYBR Green I, Taqman, Hybridisation Probes,
Molecular Beacon); map primers/probes onto different transcript variants
Nucleic Acids Research 2003, 2006 (update)
62
Primers/probes experimentally validated; search with gene name/symbol,
Entrez/Ensembl Gene identifier, SNP ID, or oligo sequence; queries can be
limited to a specific application (gene expression, DNA copy number, SNP
detection, mutation analysis, fusion gene) or organisms (20); results linked to
PubMed record and BLAST; frequently updated;
Gene expression assay viewer requires Adobe SVG viewer plug-in, and only
available for human, mouse, rat.
In OBRC; 3845 real-time PCR assays for 2373 genes as of Nov. 2007.
4.8 out of 5
Web Site:
http://medgen.ugent.be/rtprimerdb/
More Info:
http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1099597360/info
Resources for real time PCR– Primer Bank
Name
Type
Key Functions
Publication Info
Times Cited
Pros
Cons
Note
YiBu’s Rating
A PCR primer bank for quantitative gene expression analysis
Web-based database
Search for pre-designed transcript-specific PCR primers for genome-scale real
time PCR assay
Nucleic Acids Research 2003
67
Searchable by GenBank Accession number, gene ID/symbol, keywords etc.;
design algorithm extensively tested and validated with success rate of 82.6%;
results output containing positions of primers and amplicons in the sequence
context of the queried gene
Only for human and mouse genes; algorithm not designed to span introns; no
graphic display;
Contains 306,800 real-time PCR primers for 33741 human genes and 27681
mouse genes as of Nov. 2007
4.8 out of 5
Web Site:
http://pga.mgh.harvard.edu/primerbank/
More Info:
http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14654707
Resources for real time PCR– qPrimerDepot
Name
Type
Key Functions
Publication Info
Times Cited
Pros
Cons
Note
YiBu’s Rating
qPrimerDepot -- a primer database for quantitative real time PCR
Web-based database
Provides optimized qRT-PCR primers for all human/mouse RefSeq genes;
NAR 2005
19
Uses GenBank RefSeq ID or gene name as input; primers are designed to
amplify desired templates under unified annealing temperature; avoids genomic
DNA contamination (for intron-bearing genes); simple user interface;
Validation results show 70-94% successful rate;
Results output without sequence context display of primer/amplicons positions
In OBRC; built on Primer3;
4.5 out of 5
Web Site for Human Genes:
http://primerdepot.nci.nih.gov/
Web Site for Mouse Genes:
http://mouseprimerdepot.nci.nih.gov/
More Info:
http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1174922412/info
Resources for real time PCR– QPPD
Name
Type
Key Functions
Publication Info
Times Cited
Pros
Cons
Note
YiBu’s Rating
QPPD -- Quantitative PCR Primer Database for human and mouse
Web-based database
Search for published quantitative/real time RT-PCR primer and probes for
studying of human and mouse gene expression.
Unpublished
n/a
All primers were experimentally validated with literature references; uses gene
name as input; searches can be limited by different assay types; output includes
graphic display of primers/amplicons positions and sizes.
Only human and mouse genes available; no user documentations and database
stats.
In OBRC
4 out of 5
Web Site:
http://web.ncifcrf.gov/rtp/GEL/primerdb/default.asp
More Info:
http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1152117830/info
Resources for real time PCR– PerlPrimer
Name
Type
Key Functions
Publication Info
Times Cited
Pros
Cons
Note
YiBu’s Rating
PerlPrimer -- cross-platform, graphical primer design for standard,
bisulphite and real-time PCR
Desktop software (for Windows, Linux and Mac OS-X)
Design primers for standard, bisulphite and real-time PCR, and sequencing.
Bioinformatics 2004
8
Cross-platform; versatile applications; retrieving sequences from Ensembl as
input; QPCR primer design without manual intron-exon boundary entry;
BLAST search primers; primer pair quality analysis; ORF and CpG island
detection;
Requires local installation of the software; automatic sequence retrieval only
through Esemble (selected eukaryotic genomes) not NCBI (more
comprehensive).
In OBRC
4 out of 5
Web Site:
http://perlprimer.sourceforge.net/index.html
PerlPrimer screenshots:
http://perlprimer.sourceforge.net/screenshots.html
More Info:
http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1167845497/info
Resources for Site-Directed Mutagenesis PCR – PrimerX
Name
Type
Key Functions
Publication Info
Times Cited
Pros
Cons
Note
YiBu’s Rating
PrimerX -- Automated design of mutagenic primers for site-directed
mutagenesis
Web-based software
Design mutagenic primers for site-directed mutagenesis.
unpublished
n/a
Uses either DNA or protein sequences as input; results can be customized for
three different commercial mutagenesis kits; straightforward user interface,
No data on evaluation and user feedback;
In OBRC
4 out of 5
Web Site:
http://www.bioinformatics.org/primerx/
More Info:
http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1175091818/info
Resources for PCR Primer or Oligo Analysis





AutoDimer
IDT OligoAnalyzer 3.0
PUNS
NCBI BLAST
UCSC In-Silico PCR
Resources for PCR Primer or Oligo Analysis
– AutoDimer
Name
Type
Key Functions
Publication Info
Times Cited
Pros
Cons
Note
YiBu’s Rating
AutoDimer -- a screening tool for primer-dimer and hairpin structures
Web-based or desktop software
Rapidly screen PCR primers for primer-dimer and hairpin interactions in short
DNA oligomers (< 30 nucleotides)
Biotechniques 2004
17
Suited for screening multiplex PCR primers; output has alignment;
Requires manually formatted primers file as input; crude web layout; the webbased version is limited 100 sequences/run and maximum oligomer length of
75 nucleotides or less.
In OBRC
4 out of 5
Web Site:
http://www.cstl.nist.gov/div831/strbase/AutoDimerHomepage/AutoDimerProgramHomepage.htm
More Info:
http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1154964478/info
Resources for PCR Primer or Oligo Analysis
–IDT OligoAnalyzer 3.0
Name
Type
Key Functions
Publication Info
Times Cited
Pros
Cons
Note
YiBu’s Rating
IDT OligoAnalyzer 3.0
Web-based software
Analyze primer/oligo sequence and structure.
Unpublished
n/a
Includes analysis of hairpins, self-dimer, heterodimer; customizable
primer/oligo and salt concentrations; many options for various sequence
modifications; direct submission for NCBI BLAST; allows direct order of
primer/oligo;
No evaluation data.
4 out of 5
Web Site:
http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/
Online Instruction:
http://www.idtdna.com/Analyzer/Applications/Instructions/Default.aspx?AnalyzerInstructions=true
Resources for PCR Primer Specificity Analysis
– PUNS
Name
Type
Key Functions
Publication Info
Times Cited
Pros
Cons
Note
YiBu’s Rating
PUNS -- Transcriptomic- and genomic-in silico PCR for enhanced primer
design
Web-based or desktop software (Windows, Linux)
Use in silico PCR to verify primer specificity by comparing the primers against
the entire transcriptome/genome and looking for alternate binding and potential
alternate amplicons. Particularly suited for the identification of highly selective
primers for quantitative microarray validation;
Bioinformatics 2004
4
Focuses on primer specificity analysis; capable of design cross-species primers;
uses pre-designed primers as input; good online documentation
For specificity check against transcriptome, works only with model organisms
whose transcriptome info are available in UniGene; requires special input file
format; unnecessary complicated interface;
in OBRC
4 out of 5
Web Site:
http://okeylabimac.med.utoronto.ca/PUNS/
More Info:
http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1175092441/info
Resources for PCR Primer Specificity Analysis
– NCBI BLAST
http://www.ncbi.nlm.nih.gov/blast/Blast.cgi?PAGE=Nucleotides&PROGRAM=blastn&MEGABLAST=on&BLAST_PROGRAMS=megaBlast&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on
Resources for PCR Primer Mapping
– UCSC In-Silico PCR
http://genome.ucsc.edu/cgi-bin/hgPcr?db=mm9
Resources for PCR Primer Mapping/Amplicon Size
– SMS Tool
http://www.bioinformatics.org/sms2/pcr_products.html
http://www.bioinformatics.org/sms2/index.html
Please evaluate this workshop to help me improving
future presentations:
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Have questions or comments about this workshop?
Please contact:
Yi-Bu Chen, Ph.D.
Bioinformatics Specialist
Norris Medical Library
University of Southern California
323-442-3309
yibuchen@belen.hsc.usc.edu
Primer Design Tools for Multiplex PCR
 MultiPLX
 PrimerStation
Primer Design Tools for Multiplex PCR–
MultiPLX
Name
Type
Key Functions
Publication Info
Times Cited
Pros
Cons
Note
YiBu’s Rating
MultiPLX -- automatic grouping and evaluation of PCR primers
Web-based or desktop software (Windows, Linux)
Automatic grouping large number of PCR primers pairs based on analysis of
primers properties and compatibilities.
Bioinformatics 2005
2
Capable of analyzing thousands of primers;
Input requires specially formatted primer file; poor online documentation;
3.5 out of 5
Web Site:
http://bioinfo.ebc.ee/multiplx/
More Info:
http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&dopt=AbstractPlus&list_uids=15598831
Primer Design Tools for Multiplex PCR–
PrimerStation
Name
Type
Key Functions
Publication Info
Times Cited
Pros
Cons
Note
YiBu’s Rating
PrimerStation -- a highly specific multiplex genomic PCR primer design
server for the human genome
Web-based software
Design highly specific and accurate multiplex genomic PCR primer for human
genome.
NAR 2006
0 (too new)
Uses multiple Genbank RefSeq ID or chromosomal coordinates as inputs;
allows exon-only amplification; options to avoid SNPs region or CA-repeats;
configurable settings for primer design;
Only for human genome;
In OBRC
4.5 out of 5
Web Site:
http://ps.cb.k.u-tokyo.ac.jp/index.html
More Info:
http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1154793164/info
Resources for Microarray Probe Design
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


NCBI Probe Database
OligoWiz 2.0
ROSO
YODA
Resources for Microarray Probe Design –OligoWiz 2.0
Name
Type
Key Functions
Publication Info
Times Cited
Pros
Cons
Note
YiBu’s Rating
OligoWiz 2.0 -- integrating sequence feature annotation into the design of
microarray probes
Web-based client-server solution
Perform intelligent design of oligonucleotides against a given set of target
sequences for DNA microarray application.
NAR 2005
8
The input sequence file can be in FASTA or annotation containing tab format;
integrated species database for reducing or eliminating cross-hybridizations; in
addition to probe selection according to a series of probe quality parameters,
cross-hybridization, Tm, position in transcript, probe folding and lowcomplexity, the program facilitates automatic placement of probes relative to
the sequence annotation; evaluation study shows consistent hybridization
results; decent online user guide;
Requires download/install Java-based Graphic User Interface
In OBRC
4 out of 5
Web Site:
http://www.cbs.dtu.dk/services/OligoWiz2/
More Info:
http://www.hsls.pitt.edu/guides/genetics/obrc/gene_expression/microarray_design_probes/URL111876
7631/info
Resources for Microarray Probe Design –ROSO
Name
Type
Key Functions
Publication Info
Times Cited
Pros
Cons
Note
YiBu’s Rating
ROSO -- Optimizing oligonucleotide probes for microarrays
Web-based software
Design optimal oligonucleotide probe sets for microarrays.
Bioinformatics 2004
23
Uses FASTA formatted file for both targeted cDNA sequences and a
facultative external file containing sequences to be avoided in crosshybridization; the facultative files are provided for each model organisms,
customized facultative can be uploaded; settings for hybridization conditions
and probe secondary structures;
No data on evaluation and user feedback;
4 out of 5
Web Site:
http://pbil.univ-lyon1.fr/roso/Home.php
More Info:
http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14734320
Resources for Microarray Probe Design –YODA
Name
Type
Key Functions
Publication Info
Times Cited
Pros
Cons
Note
YiBu’s Rating
YODA -- selecting signature oligonucleotides
Desktop software
Select signature sequences for microarray and other applications
Bioinformatics 2004
15
Cross-platform (Windows, Mac OS X, Linux); relies on a custom sequence
similarity search algorithm instead of BLAST to minimize false negatives;
supports multiple probe design goals including single-genome, multiplegenome, pathogen-host and species/strain-identification; many configurable
parameters; uses FASTA formatted files for both targeted and avoided
sequences.
Requires download and installation on local PC; no data on evaluation and user
feedback;
4 out of 5
Web Site:
http://pathport.vbi.vt.edu/YODA/
More Info:
http://bioinformatics.oxfordjournals.org/cgi/content/full/21/8/1365
PCR Primer Design Resources for SNPs and Genotyping Purposes




NCBI Probe Database
PrimerZ
MuPlex
SNPBox
Public PCR Primers/Oligo Probes Repository
– The NCBI Probe Database
Name
Type
Key Functions
Publication Info
Times Cited
Pros
Cons
Note
YiBu’s Rating
NCBI Probe Database
Web-based database
Search for documented PCR primers and oligo probes for genotyping, gene
expression, SNP discovery, gene silencing and genome mapping applications.
Unpublished
n/a
The largest database of its kind; results including information on reagent
distributors, probe effectiveness, and computed sequence similarities; Entrez
allows search to be limited by applications, probe type, and model organism;
n/a
4.5 out of 5
Web Site:
Database Overview:
http://www.ncbi.nlm.nih.gov/genome/probe/doc/Overview.shtml
Database Query Tips:
http://www.ncbi.nlm.nih.gov/genome/probe/doc/QueryTips.shtml
PCR Primer Design Resources for SNPs and Genotyping Purposes
– PrimerZ
Name
Type
Key Functions
Publication Info
Times Cited
Pros
Cons
Note
YiBu’s Rating
PrimerZ -- streamlined primer design for promoters, exons and
human/mouse SNPs
Web-based software
Design PCR primers for promoters, exons, and human/mouse SNPs
Nucleic Acid Research 2007
0 (too new)
Uses gene name, Ensembl ID, rs# as input; settings for amplicon region and
length, as well as PCR product sizes; allow batch rsSNP processing; frequently
updated; offers many advance design settings; interactive results output
reported successful rate over 70%.
built on Primer3; in OBRC.
4.5 out of 5
Web Site:
http://genepipe.ngc.sinica.edu.tw/primerz/beginDesign.do
More Info:
http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1190992855/info
PCR Primer Design Tools for SNPs and Genotyping Purposes–
MuPlex
Name
Type
Key Functions
Publication Info
Times Cited
Pros
Cons
Note
YiBu’s Rating
MuPlex -- multi-objective multiplex PCR assay design
Web-based software
Design primers for high-throughput multiplex PCR assays, including
genotyping.
NAR 2005
7
Input uses FASTA sequence/file; sequence with SNP brackets; allows masked
region; results of assay solutions emailed; many design options;
No evaluation data
In OBRC; build on Primer3
4 out of 5
Web Site:
http://genomics14.bu.edu:8080/MuPlex/MuPlex.html
More Info:
http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1135006767/info
PCR Primer Design Tools for SNPs and Genotyping Purposes–
SNPBox
Name
Type
Key Functions
Publication Info
Times Cited
Pros
Cons
Note
YiBu’s Rating
SNPbox: a modular software package for large-scale primer design
Web-based software
Design PCR primers for large-scale amplification and sequencing projects
aimed at constructing single nucleotide polymorphisms maps; design of primer
sets for mutation analysis, STR marker genotyping and microarray oligos.
NAR 2004/Bioinformatics 2005
5
Uses GenBank ID or FASTA sequence file as input; no prior knowledge of
rsID needed; many design options; offers SNP; Exon and Saturation module;
Interface not well designed; inconsistent server processing speed; requires
Adobe SVG viewer web browser plug-in for interactive results visualization.
In OBRC; build on Primer3;
3.5 out of 5
Web Site:
http://www.snpbox.org/
More Info:
http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1097782408/info
Primer Design Tools for Degenerate PCR
 Primaclade
 GeneFisher2
 CODEHOP
Primer Design Tools for Degenerate PCR– Primaclade
Name
Type
Key Functions
Publication Info
Times Cited
Pros
Cons
Note
YiBu’s Rating
Primaclade -- a flexible tool to find conserved PCR primers across
multiple species
Web-based software
Design degenerated PCR primers based on conserved regions from a multiple
species nucleotide alignment file
Bioinformatics 2005
9
Uses a multiple species nucleotide alignment file as input; accepts several
alignment format;
May not work well when sequences in the alignment are > 29% divergent; does
not use IUPAC codes in the input alignment; last updated in 2004
Built on Primer3; in OBRC
4 out of 5
Web Site:
http://www.umsl.edu/services/kellogg/primaclade.html
More Info:
http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1167846864/info
Primer Design Tools for Degenerate PCR– GeneFisher2
Name
Type
Key Functions
Publication Info
Times Cited
Pros
Cons
Note
YiBu’s Rating
GeneFisher2 – an interactive tool for designing degenerate PCR primers
flexible tool to find conserved PCR primers across multiple species
Web-based software
Design degenerated PCR primers based on conserved regions among multiple
DNA or protein sequences
Proc Int Conf Intell Syst Mol Biol. 1996
38
Uses but does not require pre-aligned sequences; integrated alignment tools to
align starting sequences; accepts both DNA and protein sequences;
Only accepts FASTA format alignment; no evaluation data;
Built on Primer3;
4 out of 5
Web Site:
http://bibiserv.techfak.uni-bielefeld.de/genefisher2/
More Info:
http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=8877506
Primer Design Tools for Degenerate PCR– CODEHOP
Name
Type
Key Functions
Publication Info
Times Cited
Pros
Cons
Note
YiBu’s Rating
CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer)
PCR primer design
Web-based software
Design degenerate PCR primers based on multiple protein sequences
alignments
Nucleic Acids Research 2003
37
Widely cited with many successful applications; settings for genetic code and
codon usage;
Requires local multiple alignment as input and must be in Blocks Database
format;
In OBRC
4 out of 5
Web Site:
http://blocks.fhcrc.org/codehop.html
More Info:
http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1118954832/info
Primer Design Resources for Methylation PCR




MethPrimer
methBLAST and methPrimerDB
BiSearch
PerlPrimer
Primer Design Resources for Methylation PCR–
MethPrimer
Name
Type
Key Functions
Publication Info
Times Cited
Pros
Cons
Note
YiBu’s Rating
MethPrimer: designing primers for methylation PCRs
Web-based software
Design primers for methylation-specific, bisulfite-sequencing PCR, or bisulfiterestriction PCR assays.
Bioinformatics 2002
123
Widely used; uses DNA sequence in any format as input; no need to modify
sequences; very simple interface; settings for primers; output includes graphics
and sequence alignment; fast processing
n/a
Build on Primer3; in OBRC;
4.5 out of 5
Web Site:
http://www.urogene.org/methprimer/
More Info:
http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1167846108/info
Primer Design Tools for Methylation PCR–
methBLAST and methPrimerDB
Name
Type
Key Functions
Publication Info
Times Cited
Pros
Cons
Note
YiBu’s Rating
methBLAST and methPrimerDB: web-tools for PCR based methylation
analysis
Web-based software
methPrimerDB for validated PCR primers for DNA methylation analysis;
methBLAST for in silico assessment of primer specificity in PCR based
methylation assays.
BMC Bioinformatics 2006
0 (too new)
Search validated primers by gene name with organism limit; in silico PCR
using bisulfite converted DNA as input;
Only 243 primer sets available in the methPrimerDB (as of Oct. 2007)
in OBRC; from the same group of MethPrimer
4 out of 5
methPrimerDB Web Site:
http://medgen.ugent.be/methprimerdb/
methBLAST Web Site:
http://medgen.ugent.be/methBLAST/
More Info:
http://www.biomedcentral.com/1471-2105/7/496
Primer Design Tools for Methylation PCR–
BiSearch
Name
Type
Key Functions
Publication Info
Times Cited
Pros
Cons
Note
YiBu’s Rating
BiSearch: primer-design and search tool for PCR on bisulfite-treated
genomes
Web-based software
Design primers for both bisulfite-treated and native DNA templates.
NAR 2005, BMC Bioinformatics 2006, Methods Mol Biol 2007
8
Uses plain text formatted sequence as input; integrated high-speed ePCR tool
for fast detection of mispriming sites and alternative PCR products in cDNA
libraries and native or bisulfite-treated genomes; tools for calculating primer
Tm and scores; configurable designing/scoring parameters; fast server;
ePCR only available for 4 native/bisufite-treated mammalian genomes.
4.5 out of 5
Web Site:
http://bisearch.enzim.hu/
More Info:
http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15653630
Primer Design Tools for Methylation PCR–
PerlPrimer
Name
Type
Key Functions
Publication Info
Times Cited
Pros
Cons
Note
YiBu’s Rating
PerlPrimer -- cross-platform, graphical primer design for standard,
bisulphite and real-time PCR
Desktop software (for Windows, Linux and Mac OS-X)
Design primers for standard, bisulphite and real-time PCR, and sequencing.
Bioinformatics 2004
8
Cross-platform; versatile applications; retrieving sequences from Ensembl as
input; QPCR primer design without manual intron-exon boundary entry;
BLAST search primers; primer pair quality analysis; ORF and CpG island
detection;
Requires local installation of the software; automatic sequence retrieval only
through Esemble (selected eukaryotic genomes) not NCBI (more
comprehensive).
In OBRC
4 out of 5
Web Site:
http://perlprimer.sourceforge.net/index.html
PerlPrimer screenshots:
http://perlprimer.sourceforge.net/screenshots.html
More Info:
http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1167845497/info
Please evaluate this workshop to help me improving
future presentations:
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Have questions or comments about this workshop?
Please contact:
Yi-Bu Chen, Ph.D.
Bioinformatics Specialist
Norris Medical Library
University of Southern California
323-442-3309
yibuchen@belen.hsc.usc.edu
Useful web sites for design degenerate PCR primers
http://boneslab.bio.ntnu.no/degpcrshortguide.htm
http://info.med.yale.edu/mbb/koelle/protocols/protocol_degenerate_PCR.html
http://www.mcb.uct.ac.za//pcroptim.htm#Degenerate
http://www.protocol-online.org/prot/Molecular_Biology/PCR/Degenerate_PCR/
http://cgat.ukm.my/protease/degpcr.html